Lateral flow immunoassay test reader and method of use

ABSTRACT

A reader for a lateral flow test device includes a tray or drawer, extendable from the reader, which receives the test device. The tray includes a calibration test pattern affixed or printed thereon placed proximate to the test device and in alignment with the axis of the test device. As the tray is closed and the test device is inserted to the reader, the calibration test pattern is first read by an optics unit including a photodiode. The resulting photodiode output provides a calibration curve S that the reader then uses to correct for any non-linear response of the reader&#39;s optical or electronic systems, thus insuring that every reader will yield the same readout for a given test cartridge, despite reader-to-reader variations or reader degradation with time. One use of the reader is for detection of SARS-CoV-2 infection.

PRIORITY

This application claims priority to U.S. Provisional application Ser.No. 63/029,003 filed on May 22, 2020, the content of which isincorporated by reference herein, including the appendix thereof. Thisapplication also claims priority as a continuation of U.S. applicationSer. No. 16/885,436 filed May 28, 2020.

FIELD

This disclosure relates to the field of readers for lateral flowimmunoassay test devices. Such test devices typically take the formfactor of a test strip or cartridge.

BACKGROUND

Lateral flow immunoassay (LFA) devices are a paper ornitrocellulose-based platform for the detection and optionalquantitation of analytes, in complex mixtures (e.g. biological fluidssuch as blood or saliva). The sample is placed in a sample well of thetest device, and the results are obtained in typically 3-30 minutes.Such test devices can exist as standalone devices, in which is thedevice is read by the unaided eye. Such test devices may also be used inconjunction with a dedicated reader. The reader includes opticalcomponents, e.g., imager or photodiode array, for assessing the teststrip and a processing unit generating a result, e.g., “positive”,“negative” or the like. LFA test devices are used in a variety oftesting scenarios, including pregnancy detection, detection of antigensindicating infection by a virus, detection of biomarkers of disease,metabolites, and other molecular targets, as well as screening foranimal diseases, chemicals, toxins, and water pollutants, among others.

Background information on lateral flow assays and related chemistry isfound in the review article of K. Koczula et al., Lateral Flow Assays,Essays in Biochemistry (2016) v 0.60 p. 111-120, the content of which isincorporated by reference herein. Dedicated readers for lateral flowimmunoassay devices are disclosed in E. Pilavaki et al., OptimizedLateral Flow Immunoassay Reader for the Detection of Infectious Diseasesin Developing Countries, Sensors vol. 17, 2673 (2017); Y. Yang et al.,Development of a Quantifiable Optical Reader for Lateral FlowImmunoassay, proceedings of the 8th International Conference onBiomedical Engineering and Informatics (BME)(2015) p. 344-349; Xie etal., U.S. Pat. No. 10,295,472; Lu et al., US patent applicationpublication 2009/0155921 and Fleming et al., US patent applicationpublication 2020/0001299.

By way of example and not limitation, and as is explained in the Koczulaet a. article, and with reference to FIGS. 1 and 2A-2B of the appendeddrawings, the principle behind a typical configuration of a LFA issimple: a liquid sample (or its extract, e.g., blood, sweat, urine,saliva etc.) containing the analyte of interest is introduced into asample well of a test device (e.g., the test strip 10). The liquidsample progresses via capillary action through various zones (typicallynarrow lines oriented perpendicular to the long axis of the strip), onwhich molecules that can interact with the analyte are attached. Atypical lateral flow test strip 10 consists of overlapping membranesthat are mounted on a backing card for better stability and handling. Asshown in FIG. 2A, the liquid sample is applied at one end of the strip10, on the absorbent sample pad, which is impregnated with buffer saltsand surfactants that make the sample suitable for interaction with thedetection system. The sample pad ensures that the analyte present in thesample will be capable of binding to the capture reagents of conjugatesand on the membrane. The treated sample migrates through the conjugaterelease pad, which contains antibodies that are specific to the targetanalyte and are conjugated to colored or fluorescent particles—mostcommonly colloidal gold and latex microspheres.

The sample, together with the conjugated antibody bound to the targetanalyte, migrates along the strip 10 into the detection zone. Thedetection zone consists of a porous membrane (usually composed ofnitrocellulose) with specific biological components (mostly antibodiesor antigens) immobilized in lines oriented perpendicular to the longaxis of the test strip. Their role is to react with the analyte bound tothe conjugated antibody (Ab). Recognition of the sample analyte resultsin an appropriate response on the test line, while a response on acontrol line indicates the proper liquid flow through the strip. Theread-out, represented by the lines appearing with different intensities(see FIG. 2B), can be assessed by naked eye or alternatively using adedicated reader, e.g., one of the readers in the above-citedreferences. In order to test multiple analytes simultaneously under thesame conditions, additional test lines of antibodies specific todifferent analytes can be immobilized in an array format. The liquidflows across the device because of the capillary force of the stripmaterial and, to maintain this movement, an absorbent pad is attached atthe end of the strip. The role of the absorbent pad is to wick theexcess reagents and prevent backflow of the liquid.

While FIGS. 1 and 2A show the test device in the form of a strip 10, itis also known to incorporate the strip 10 into a cartridge-type deviceas shown in FIG. 2 of US '472 patent cited above or the Lu et al. patentapplication publication, FIG. 7 thereof.

The present reader and related methods of this disclosure are designedto be used in conjunction with a test device in the form factor of acartridge containing a test strip; however the teachings below can beadapted to a test strip format per se, such as shown in FIGS. 1 and 2 ofthis document and thus the present disclosure is intended to cover bothtypes of test devices.

Additionally, while the present disclosure is capable of being used inconjunction with any of the currently known purposes of lateral flowimmunoassays (as explained above in the previously cited patents andtechnical literature, such descriptions of which are incorporated byreference), one particular application of the present disclosure is areader for a lateral flow immunoassay test device configured fordetection of Immunoglobulin M (IgM) and Immunoglobulin G (IgG)antibodies in human serum, whole blood or plasma from individualssuspected of infection with the SARS-CoV-2 virus, which causes a diseasereferred to as COVID-19. SARS-CoV-2 is the name assigned to the novelcoronavirus currently causing a worldwide pandemic.

SUMMARY

In a first aspect of this disclosure, a reader is provided for a lateralflow test device having an axis and one or more test lines and a controlline oriented perpendicular to the axis. The reader includes a housingenclosing electronics and an optics unit configured for reading the oneor more test lines and the control line of the test device. The readerfurther includes a tray extendable from the housing between a closedposition and an open position. The tray is adapted to receive the testdevice when the tray is extended to the open position, and when the testdevice is placed in the tray and the tray moved to a closed position theone or more test lines and control line are read by the optics unit. Thetray further includes an upper surface, the upper surface provided withan optical calibration test pattern spaced from the test device andpositioned in alignment with the axis of the test device. The testpattern facilitates performance of a self-test of the optics unit uponmovement of the tray from the open position to the closed position andimmediately before the test device is read.

In one possible format, the calibration test pattern is in the form of alinear series of bands of known, graded optical intensities, each of thebands oriented perpendicular to the axis of the test device. For examplethere may be at least five or at least ten bands of known graded opticalintensities. In one possible configuration, the calibration test patternis printed on a material and the material is placed in a designatedlocation on the upper surface of the tray.

In one embodiment, the optics unit includes a diode laser generating alight output, a line generator converting the light output of the diodelaser to a line format, and a photodiode reading the intensity of thelaser light reflected from the test device. The line format of the laserlight is oriented perpendicular to the axis of the test device and inthe same orientation of each of the bands of the calibration opticaltest pattern.

The reader may include several features, such as a rechargeable batteryand a port receiving a recharging cable for recharging the battery, andRFID reader, and/or a wireless transmitter transmitting results of areading operation to a remote computing device.

In one possible configuration the test device is in the form of a teststrip. Alternatively, the test device can take the form of a cartridgecontaining a test strip.

In one possible use, the test device includes a test strip configured totest for presence of antibodies to the SARS-CoV-2 virus. A testingsystem is disclosed including the test device configured to test forpresence of antibodies to the SARS-CoV-2 virus and the reader of thisdisclosure.

In another aspect, a method is provided for reading a lateral flow testdevice having one or more test lines and a control line with a readerhaving an electro-optical system (electronics and an optics unit). Thereader includes a tray extendable from the reader from an open positionin which the test device is placed in the tray to a closed positionwhereby the test device is read. The method includes the steps of:reading a calibration test pattern placed on the tray with the opticsunit of the reader, the optics unit including a photodiode; performing aself-test of the reader's electro-optical system, wherein the self-testincludes generating a calibration curve S characterizing any non-linearresponse of the components of the electro-optical system; and readingthe test and control lines of the test device with the optics unit,including using the calibration curve S to convert photodiode outputvalues to % absorbance values.

In another aspect, a method is provided for calibrating a reader for alateral flow test device. The reader includes an optics unit including aphotodiode receiving light reflected from a test strip provided with thetest device. The method includes steps of: a) scanning in the reader acalibration test pattern; b) acquiring photodiode output values duringthe scanning step a); c) optionally inverting the output values; d)calculating average peak heights Ai in the output values; e) storing theresults of step d) as an array of peak heights and linear position data[Ai, Pi], the linear position data Pi indicative of incremental linearpositions at which the calibration test pattern was scanned in step a);and f) fitting a spline curve S to the array data and saving theequations for the spline curve S, wherein the spine curve Scharacterizes any non-linear response of the optics system or associatedelectronics.

In the above method, in one configuration the scanning step a) comprisesmanual insertion of a tray carrying the test device into the reader, andwherein the tray includes the calibration test pattern. The test deviceincludes a test strip configured to test for presence of antibodies tothe SARS-CoV-2 virus.

In still another aspect, a method of reading a lateral flow test devicewith a reader is disclosed. The reader including an optics unitincluding a photodiode receiving light reflected from a test stripprovided with the test device. The method includes the steps of: a)scanning with the reader the test strip; b) acquiring photodiode outputvalues during the scanning step a); c) optionally inverting the outputvalues; d) converting the photodiode output values to % absorbancevalues using equations for a spline curve S, wherein the spine curve Scharacterizes any non-linear response of the optics system, and whereinspline curve S is generated during calibration of the optics unit priorto scanning step a); e) identifying peaks in normalized absorbance data;f) assigning labels to the peaks identified in step e); g) applyinglocal baseline corrections and calculating peak areas for each of thelabeled peaks of step f); and h) calculating ratios of peak areasassociated with test lines of the test device to a control line of thetest device.

In still another aspect, a tray is provided for holding a lateral flowtest device and configured for movement between open and insertedpositions relative to a reader for reading the test device, the readerfurther including a ratchet pawl and a spring. The tray includes asurface having features for receiving and holding the test device; a setof ratchet teeth formed on the tray, wherein the pawl and spring of thereader are positioned so as to bias the pawl into engagement with theratchet teeth; the tray further includes a first feature disengaging thepawl from the set of ratchet teeth upon complete insertion of the trayinto the reader and enabling the tray to be withdrawn from the reader;wherein when the tray is in intermediate positions between the open andinserted position the pawl, spring and ratchet teeth prevent retractionof the tray from the reader.

In another possible configuration, QR codes are placed on the bottomsurface of the cartridge which are read by an accessory device on whichthe reader sits. The accessory device provides a means for moving thetray in and out with the aid of a threaded rod in a programmed fashion.This configuration allows for reading the elapsed time for emergence ofa positive result on the test strip, which can provide furtherinformation useful in assessing the state of the sample and thereforeresult generated by the reader.

Accordingly, in one further aspect, an accessory unit to lateral flowassay reader having a moveable tray holding a test device is disclosed.The tray is moveable from open and closed positions. The accessory unitincludes a structure holding the reader and an electro-mechanical systemengaging the tray and moving the tray and test device into the readerbetween the open and closed positions in a programmed manner, such thatthe test device is positioned proximate to the optics unit, such that atleast one of the one or more test lines is read repeatedly for a periodof time by the optics unit, whereby the reader is able to record, eitherdirectly or indirectly, changes in the color intensity of the one ormore test lines over time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a typical configuration of a lateralflow immunoassay (LFA) test strip. The test strip is usually composed ofthe following elements: sample pad, conjugate release pad, membrane withimmobilized antibodies and an adsorbent pad. The components of the stripare usually fixed to an inert backing material

FIG. 2A is a schematic drawing showing the operation of a LFA in threesteps. Top: the sample is deposited on the sample pad and migratestowards the conjugate. Middle: the conjugated antibodies bind the targetanalyte. Bottom: migration to the test line, where the bound targetanalyte is captured, and the control line. The most commonly used LFA isthe pregnancy test (One Step hCG Urine Test), which uses hCG strips.Possible results and interpretation of the test are shown in FIG. 2B. Inthe case of a weak positive result in a pregnancy test, it isrecommended to repeat the test 1 week later.

FIG. 3 is a perspective view of a test device and optical reader of thisdisclosure.

FIG. 3A is a plan view of the test device of FIG. 3.

FIG. 4 is a more detailed schematic view of the optics of the reader ofFIG. 3.

FIG. 5 is an illustration of an optical calibration test pattern that isincorporated into the tray receiving the test device of FIG. 3.

FIG. 6 is a plot of photodiode output as a function of test patternintensities in accordance with the calibration pattern of FIG. 5,showing how the electronics in the reader can correct the photodiodeoutput signals to match the calibration pattern.

FIG. 7A is an example of a plot of the photodiode output as a functionof scan position for a given test device.

FIG. 7B is an example of the plot of FIG. 7A after signal correctionprocess has been performed.

FIG. 8 is a flowchart showing a sequence of processing operationsperformed within the reader in a self-test or calibration procedure.

FIG. 9 is a flowchart showing a sequence of processing operationsperformed within the reader during the operation of reading the teststrip within the cartridge.

FIG. 10 is an exploded view of the reader of FIG. 3 as seen inperspective view from above.

FIG. 11 is an exploded view of the reader of FIG. 3 as seen inperspective view from below.

FIG. 12 is a perspective view of the tray of FIG. 3 shown isolated fromthe reader and showing the test calibration pattern of FIG. 5 and aposition encoder pattern on the top surface of the tray.

FIG. 13 is a plan view of the tray.

FIG. 14 is a side view of the tray.

FIG. 15 is a bottom plan view of the tray; in one possible configurationa QR code is placed on the bottom which is read in an accessory device,however the QR code is not shown in FIG. 15.

FIG. 16 is a perspective view of the tray from below.

FIG. 17 is an end view of the reader showing a USB port receiving acable for recharging a battery in the reader, and an on-off switch.

FIG. 18 is a side view of the reader.

FIG. 19 is an end view of the reader showing the tray closed against thereader.

FIG. 20 is a plan view of the reader.

FIG. 21 is a cross-section through the reader along the lines 21-21 ofFIG. 20.

FIG. 22 is a cross-section through the reader with the tray carrying atest strip and the tray partially inserted into the reader to a positionwhere the calibration pattern is read by the optics of FIG. 4.

FIG. 23 is a cross-section through the reader with the tray carrying atest strip and the tray more fully inserted into the reader as comparedto FIG. 22 to a position where the control line of the test device 20 isbeing read by the optics in the reader.

FIG. 24 is a cross-section through the reader with the tray insertedinto the reader in roughly the same position as compared to FIG. 23,showing the reading of the position encoding pattern affixed to thetray, as shown in FIG. 12. The test device is omitted in this view.

FIG. 25 is cross-section through the reader showing the tray partiallywithdrawn from the reader. The test device is omitted in this view.

FIG. 26 is a plan view of the optics unit in the reader.

FIG. 27 is a cross-section through the optics unit of FIG. 26 takenalong the lines 27-27 of FIG. 26.

FIG. 28 is cross-section through the reader and tray showing theoptional ratchet and pawl mechanism preventing withdrawal of the trayprior to being fully inserted into the reader.

FIG. 29 is a perspective view of the electronics board of the readershowing more detail the photodiode and LED assemblies which are used forreading the position encoding strip on the tray during insertion of thetray into the reader.

FIG. 30 is a perspective view of an accessory unit for the reader ofFIG. 3 which includes an electro-mechanical system moving the tray intoand out of the reader.

FIG. 31 is a perspective view of the accessory unit for the readerholding the reader, with the tray in the extended position with the testcartridge loaded in the tray.

FIG. 31A is a top view of the reader, tray and test cartridge in theposition shown in FIG. 31.

FIG. 32 is a perspective view of the accessory unit for the reader withthe tray in the closed position for reading of the test cartridge.

FIG. 32A is a top view of the reader and tray in the position shown inFIG. 32.

FIG. 32B is a perspective view of the reader and accessory unit of FIG.32.

FIG. 33 is a perspective view of the electro-mechanical system designedto move the tray relative to the reader optics in a programmed manner,with the skin or housing for the accessory unit shown in dashed lines.

FIG. 34 is a perspective view of the electro-mechanical system of FIG.33 with the pull extended.

FIG. 34A is an exploded view of the electro-mechanical system of FIGS.33 and 34.

FIG. 35 is a bottom perspective view of the reader with the trayextended and showing a QR code on the test device visible through anaperture in the tray.

FIG. 36 is bottom perspective view of the reader with the tray closed.

FIG. 37 is a view of the accessory unit from below, showing theengagement of the pull component of the electro-mechanical systemengaging the tray.

FIG. 38 is a view of the accessory unit from below, showing theelectro-mechanical system moving the tray to the closed position.

In FIGS. 33, 34, 37 and 38 the housing or “skin” for the accessory unitis shown in broken lines in order to show the electro-mechanical systemand its operation in better detail.

FIG. 39 is a perspective view of the test cartridge and a guide as seenfrom above; the guide aids in placement of a QR code on the bottomsurface of the test cartridge.

FIG. 40 is a perspective view of the test cartridge and guide of FIG. 39as seen from below.

FIG. 40A is a perspective view of the test cartridge after the guide ofFIGS. 38 and 39 has been used adhere the QR code to the bottom of thetest cartridge.

FIG. 41 is an illustration of a portion of a CSV file includingidentification data and test result data generated in the reader andtransmitted wirelessly to a remote computing device, e.g., smartphone.

FIG. 42 is an illustration of a portion of a spreadsheet or spreadsheetwhich aggregates anonymized CSV files, such as the type shown in FIG.41, and arranges them in a format for data mining, generation ofreports, and other uses.

FIG. 43 is an illustration of a map showing the geographic distributionof epidemiological information based on the information in the databaseof FIG. 42.

DETAILED DESCRIPTION

Overview

As noted above, today's lateral flow immunoassays (LFAs) are notoriouslychallenging for untrained users to properly read directly by eye,especially when either manufacturing variances or the biologicalvariability common among patients leads to weakly stained test bands,posing a possibility of incorrect reads. Such scenarios can cause theuser to generate either false-negative or false-positive test results.Incorrect reads (and the false results they can generate) not onlyreflect badly on the product, but can create a public health hazard.Additionally, in the large-scale screening environment (such asemployer-sponsored screens of employees), by-hand record keeping slowstest administration workflow and introduces the very real potential forrecordkeeping errors.

The reader of this disclosure is designed to replace direct reading of atest device by eye with a more accurate and automated reading done bymachine, i.e., the reader of this disclosure. The reader can beconfigured to employ features expected to be particularly important toemployers, who are managing the safe return of employees in the currentCOVID-19 epidemic, and for FDA approved home use. A number of featuresand benefits of the reader are described at the end of this document.Such advantages and benefits will be more completely appreciated afterconsideration of the many mechanical, optical, electrical and softwareaspects that are incorporated in the design and described in thefollowing discussion with reference to FIG. 3 et seq.

In the following discussion, and with reference to FIGS. 3 and 3A, wewill discuss for purposes of illustration and not limitation a testdevice 20 having a test strip 110, the device 20 in the form of acartridge having a well 22 for introduction of the liquid sample and anopening 24 revealing a control line 26 and two test lines 28 (such asIgG and IgM lines) found on a test strip within the cartridge. The testdevice 20 is designed for insertion into a reader shown at 30, whichincludes a tray 32 or drawer that slides out from the reader 30 and hasfeatures for placement of the test device 20 into the tray andsubsequent insertion into the reader for reading. The top of the reader30 has a display 34 configured to display results from the readoperation, as will be described in detail below. The reader 30 may alsoinclude a text panel 36 for printed instructions on use or explanationsfor interpretation of the test results. The reader 30 has a rechargeablebattery and designed to be held in the hand of a user.

As an overview, and with reference to FIG. 4, the core of the reader 30itself is a simple yet precise optical system 100 employing inexpensiveoptical components: a low-power green diode laser 102 (the lightsource), a line-generator 104 which functions to convert the laser'sGaussian dot-like output into a line format 105 of laser light, and aphotodiode 106, to read the intensity of the laser light reflected fromthe test strip 110 contained within the cartridge 20 of FIG. 3. Thephotodiode in one embodiment has peak response for incident wavelengthof 525 nm, which is at or close to the wavelength of light emitted bythe laser. The line generator 104 may take the form of a lens orcombination of a lens and slits, masks, and/or baffles shaping andconverting the light output into a line. The design further includes ahalf silvered mirror or beam splitter 112 which directs the incidentline of laser light on the test strip and allows the reflected light topass through the mirror 112 and fall on the photodiode 106. The apertureor field of view of the photodiode is preferably large enough so as toencompass the ray bundle reflected from the test strip 110, e.g., 4 mmin diameter.

The laser line 105, projected onto the test strip as a line parallel tothe cartridge's test and control lines (see FIG. 5), is very narrow(e.g., about 48 μm wide), permitting high-resolution reads (enablingdiscrimination of lines from stains) without an expensive camera andlenses, plus the ability to perform boxcar averaging of the scan, e.g.,to optimize the optical system's signal-to-noise (S/N) ratio, for thebest possible sensitivity and dynamic range.

Scanning the laser line along the length of the test strip isaccomplished by the manual motion of inserting a tray 32 (FIG. 3)carrying the test device 20 into the reader 30 by the user (oroptionally by withdrawal of the tray carrying the test device from thereader, or during insertion and withdrawal), as indicated by the arrows113 (FIG. 4), thus enabling a reader with no powered moving parts. Forexample, in one possible configuration, the reader does not require theuse of a motorized stage or other mechanical system to move the opticsor test strip relative to each other in a read operation. The reader mayinclude a separate system, such as an optical system in the form of LEDSand one or more photodiodes, to determine the position of the cartridgeand test strip within the reader during read operations, which will bedescribed later.

When the reader is not in use, the optical components are in an OFFstate and the compartment containing the optical components is closedfrom the environment by the tray 32 (FIG. 3) when it is in a closed orstowed position, thereby protecting the optical components from dust,dirt, and moisture. The tray 32 is retracted from the reader 20 by theuser so as to enable insertion of a test cartridge 20 into the tray forreading and this operation turns the laser light source and relatedoptical components into an ON state and ready for read operations. Theseoperations will be described in detail later in this document.

An optical calibration test pattern 200 (see FIG. 5) is affixed on thetop surface of the tray 32 in alignment with the long axis of the teststrip within the cartridge 20 as shown in FIG. 13. This test pattern canbe printed in a controlled manner and affixed as a sticker or insert ata designated location on the tray, see the description below. After thetest cartridge 20 is installed in the tray and the tray 32 inserted intothe reader, this calibration test pattern enables the reader to performa sophisticated self-test immediately before the test strip of thecartridge itself is read, described in detail in conjunction with FIG. 8later in this document. The calibration test pattern (in top view, asviewed by the photodiode) is shown in FIG. 5 and takes the form of amultitude of parallel bands 202 (say 5, or more, or 10 or more, such as13 or 20) of gradually varying intensity or darkness as shown in FIG. 5.Such bands 202 may be gray-scale or colored, e.g., colored red. Themanner of conducting this calibration is described below. Briefly, inone possible configuration, the pattern consists of 13 parallel redlines in a linear series of known, graded intensities from 0.05 to 1.0with 1.0 being maximum intensity. The lines are oriented in the samedirection as the projection of the laser light line 105 onto the teststrip. The movement of the tray out of the reader to load the cartridgeturns on the reader, which then reads the 13-line test pattern as thepattern moves past the photodiode and just ahead of the insertedcartridge as the cartridge is inserted into the reader. The resultingphotodiode output provides a calibration curve that the reader then usesto correct for any non-linear response of the reader's optical orelectronic systems, thus insuring that every reader will yield the samereadout for a given test cartridge, despite reader-to-reader variationsor reader degradation with time. This correction of the test patternintensity is shown in FIG. 6.

Immediately following the test pattern scan the test cartridge isscanned, yielding a readout like that in FIG. 7A. FIG. 7B shows the samedata with a 3-point moving average applied to reduce noise.

During signal processing (see FIG. 9, described below) a peak-finderalgorithm identifies the control, and test line peaks, such as IgM, andIgG peaks (left to right in the above example, respectively), ifpresent, based on their expected positions within pre-defined scanposition ranges. The areas under these peaks (AUPs) are calculated,including a baseline background subtraction step, and finally the ratiosof the IgM and IgG AUPs to that of the control line AUP are calculated.A line is considered ‘present’ if and only if its peak is identified bythe peak-finder algorithm within its expected range of positions, itspeak height exceeds an arbitrary pre-set multiple of the baseline noise,and its width at half-height falls within a pre-set range of values.

The results of the test can be reported directly to the user/testadministrator by means of a display 34 (FIG. 3) incorporated on thesurface of the reader. While a variety of possible reporting mechanismsare contemplated, such as “positive”, “negative” or the like, in onepossible configuration the results are reported as an alphanumericindicia, such as “1”, “2”, “3”, “4”, or “5”, “A”, “B” (or some otherrange of integers or letters), with the number or letter correspondingto a particular interpretation of the test cartridge. The manner ofhuman interpretation of the test result number, e.g. “4”, can be done byreference to literature accompanying the reader, by means of a table orexplanatory text printed on the reader itself, or in other format. Anexample of literature for interpretation of the test report number for aCOVID-19 test embodiment of the reader is found at the Appendix of ourprior provisional. It will be understood that different types of testswill have different interpretation of the scores and the exampleprovided is just one of myriad possible ways in which the test resultcan be reported to the user. The reporting a number as described aboveis therefor offered by way of example and not limitation.

With the above description in mind, a more detailed description of theillustrated embodiment will now be set forth. The presently preferredembodiments are offered by way of example and not limitation.

Test Cartridge and Sample Collection

One form of the test device suitable for use with the reader 30 is shownin FIG. 3 and in plan view of FIG. 3A. The test device is in the form ofa cartridge 20 or carrier which holds within it a test strip 110 havingone or more test lines and control line shown at 26 and 28 in FIG. 3.The cartridge 20 includes an aperture or opening 24 which reveals thecontrol and test lines enabling such lines to be read by the reader. Asample well port 22 is provided for introduction of a fluid sample(e.g., blood, saliva, etc.) which then is able to migrate through thetest strip as explained in conjunction with FIGS. 1 and 2. The testdevice 20 form factor is not particularly important. The test deviceincludes a long or major axis A coincident with the long axis of thetest strip within; the test and control lines are oriented perpendicularto this axis as shown in FIG. 3A.

Reader

The reader 30 of FIG. 3 is shown in exploded view in FIG. 10. Itincludes upper and lower housings 1022A and 1022B, which may be made ofmolded plastic. The housings enclose a rechargeable lithium ion battery,electronics mounted to a circuit board 1026, including an optics unit1028 (shown schematically in FIG. 4). The electronics include aprogrammable microprocessor, memory, A/D converter, and otherconventional components, the details of which are not particularlyimportant, or will be described in detail later. The reader furtherincludes a laser diode light shield door 1030 which deploys (e.g.,swings down, using a spring, not shown) when the tray 32 is moved fromclosed to open positions thereby preventing escape of laser light fromthe interior of the reader. The light shield door could also bepositioned immediately adjacent to the aperture 1102 (FIG. 11) in theboard for the light beam, and which slides into and out of positionsobstructing the aperture and thus blocking the beam of laser light.Activation of the door could be triggered by a ramp or other featureformed in the tray 32.

The reader further includes a ratchet pawl extension spring 1032, and aratchet pawl 1034 which cooperate with a set of ratchet teeth (1502)formed in the tray 32 and shown in FIGS. 15 and 16. When the tray is inthe fully withdrawn position the head of the pawl 1034 engages with thefirst tooth of the ratchet teeth 1502 feature; as the tray is advancedthe spring pulls the pawl such that the pawl continues to engage eachtooth of the ratchet. When the tray is fully inserted, a ramp 2802(FIGS. 15, 28) moves the head of the pawl out of engagement with theratchet teeth thereby allowing the tray to be withdrawn from the reader.When the tray is fully withdrawn a second ramp 2804 moves the head ofthe pawl back into position to engage the first tooth of the ratchetteeth 1502. Thus, the ratchet feature, pawl and spring cooperate toprevent in-and-out insertion of the tray until the tray is fullyinserted, thereby insuring that both the calibration test pattern andall of the test and control lines of the test device are read.

The ratchet, pawl and spring are considered optional and they may not benecessary or provided in the tray and reader in one possibleconfiguration.

The rear of the reader (FIG. 17) includes a charging port 1702 forconnection to a cable carrying power to recharge the lithium ion batteryin the reader. This charging port could for example take the form of aUSB port. An on-off switch 1704 is also provided to turn the reader onand off.

The reader housing includes a series of raised and indented portions orgrips 2002 (FIG. 20) on the sides thereof to facilitate holding of thereader with one hand.

A. Tray and Test Cartridge Positioning

Referring to FIGS. 3 and 10-22, the tray 32 is extendable from thereader housing between closed and open positions; in the open positionthe tray receives the test device 20 as shown in FIGS. 3 and 22. Thetray 32 includes a ramp 1006 (FIG. 10) which activates limit switches2400 and 2402 mounted to the electronics board 1026 to turn on and offthe optics. In particular, as the tray is inserted into the reader, whenthe leading edge of the ramp 1006 advances within the reader andcontacts the rear limit switch 2400 (FIGS. 11, 22), the laser diodepower is activated. During withdrawal of the tray, when the leading edgeof the ramp 1006 contacts the front limit switch 2402 (FIGS. 11, 22),the laser power is disabled.

Referring to FIG. 10, the tray includes a depression or cavity 1002which has dimensions and form factor to receive snugly the test device20. The test device is inserted such that the upper surface thereof isretained snugly by two cleats 1004. The upper surface 33 of the tray 32includes an area 1008 which receives the calibration test pattern 200 ofFIG. 5. The test pattern can take the form of a printed sticker or cardwith adhesive backing printed with the graduated bands as shown in FIG.5. This area 1008, which may a slight depression molded in the topsurface 33 of the tray, facilitates placement of the calibration testpattern 200 in alignment with the axis A of the test strip and thecartridge 20 (FIGS. 3A, 12). The tray further includes a raised mount orplatform 1020 to which is affixed an optical position encoder or pattern1010 which is used for correlating test readings with linear positionsduring read operations, as explained below.

As shown in FIG. 11, the board 1026 includes an aperture 1102 thereinwhich allows the narrow line of laser light to pass through the boardand onto the calibration test pattern and test strip and thus enablingreading of both.

B. Tray Position Detection in Reader

Detection of the position of the tray 32 (and associated test cartridge20) is facilitated by the optical encoder 1010 of FIG. 10 and associatedreader assemblies for the optical encoder. These reader assemblies 1110Aand 1110B are shown in FIGS. 11 and 29; each of which includes aphotodiode 2904 and two LEDs 2902. These photodiodes are positionedoff-center and in alignment with the encoder 1010 placed on the topsurface of the tray such that as the tray is inserted into the readerthe encoder is illuminated by the LEDs 2902 and the reflectance from theencoder 1010 is read by the associated photodiode 2904. The platform onwhich the encoder 1010 is affixed serves to raise up the encoder to anelevation immediately below the photodiode such that the field of viewof the photodiode encompasses a single stripe or band of the encoder1010.

C. Optics Unit

The optics unit 1028 (FIGS. 10, 22) is shown schematically and operationthereof is discussed previously in conjunction with FIG. 4. The unit1028 is shown isolated in plan view in FIG. 26 and cross-section in FIG.27. The unit 1028 includes the laser diode light source 102, a lens 104converting the Gaussian dot output of the laser diode into a line format105 (e.g., 2 mm by 50 μm), with optional use of baffles or other beamshaping elements in the unit 1028. As shown in FIGS. 4 and 27, lightreflected from the test strip and calibration pattern passes through thehalf-slivered mirror 112 and impinges on a (single) photodetector 106.Heat sinks 2602 dissipate excess heat from the photodetector 106.

The laser diode light source 102 has an output wavelength that isoptimized for the chemistry of the test strip and test underconsideration, e.g., the size and characteristics of the particles(e.g., gold nanoparticles) that bind to antigens or other analytes inthe test sample. An output wavelength in the range of 520-540 nm is onepresently preferred embodiment of the reader and test device, in whichthe IgG and IgM antibodies of the SARS-Cov-2 virus bind to colloidalgold-labeled SARS-CoV-2 antigen on the test lines of the test strip.

D. Calibration

FIG. 8 is a flowchart showing a sequence of processing operations 800performed within the reader in a self-test or calibration procedure. Theprocedure of FIG. 8 is performed as the tray 32 containing the testcartridge 20 is manually inserted into the reader 30 and as thecalibration test pattern 200 of FIG. 5 placed on the top of the traymoves past the photodiode detector position within the reader. See FIG.22.

At step 802, a 1×N array of photodiode output values is acquired as afunction of scan position along the test pattern.

At step 804, an optional step is performed of inverting the array'svalues by dividing 1 by each data value (1/datum). This step is forconceptual simplification only; it converts negative-going valleys inthe photodiode output (corresponding to red test pattern bands orstripes) to positive-going peaks.

At step 806, the processing identifies the 13 rectangular pulses in thedata (corresponding to the 13 bands in the calibration pattern) andcalculates their average heights, by applying an amplitudediscrimination (rather than differentiation) peak-finder algorithm. Animplementation of such a pulse-finder, in Matlab format, is availableonline at https://terpconnect.umd.edu/˜toh/spectrum/findsquarepulse.m,but of course other procedures could be used.

At step 808, the processing stores the result of Step 804 as a 14-memberarray, [A_(i), P_(i)], where i=1 . . . 14, and where

A_(i)=−(% absorbance) of the ith test pattern line, as printed (14thmember=test pattern background), and

P_(i)=average peak height of the ith test pattern line's photometricread.

At step 810, a check is made to see if an error condition is present.Failure to identify all 13 peaks is considered a reader error condition,as is also the case if any or all peak heights are outside of anallowable range (empirically pre-determined, and the associatedparameter stored in memory of the reader). If an error condition ispresent the processing proceeds to block 812 and an error condition isreported on the reader display.

If no error condition is present, the processing proceeds to block 814.In this step, a cubic spline curve S with 14 “knots” and ‘naturalspline’ boundary conditions, see FIG. 6, is fitted to the data of thearray of step 808. An example of the calculation of the cubic splinecurve is at https://timodenk.com/blog/cubic-spline-interpolation/. Thesystem of equations defining the spline curve will be used tointerpolate normalized absorbance values for the photometric data fromthe cartridge scan in the Cartridge Data Processing of FIG. 9, below.Thus, the equations for the cubic spline curve S are saved in memory ofthe reader. In essence the spline curve S acts to correct the photodiodereadings for any nonlinearity in the reader optics or electronics.

E. Reading Operation and Related Calculations

As the cartridge is inserted into the reader past the point where thecalibration pattern (FIG. 5) read by the photodiode (and the abovecalibration is performed), the area of the test strip with the controland test lines is then in position for reading. See FIG. 23. At thispoint, the cartridge data processing operations 900 of FIG. 9 areperformed.

In particular, step 902 is Cartridge Data Acquisition: Acquire a 1×Narray of photodiode output values as a function of scan position alongthe cartridge's nitrocellulose strip 110 (FIG. 3, 4).

At step 904, an optional step is to invert these values by dividing 1 byeach data value (1/datum). This step is for conceptual simplificationonly; it converts negative-going valleys in the photodiode output(corresponding to positive test lines and the control line) topositive-going peaks.

At step 906, using the system of equations describing the spline curve Sfitted to the test-pattern data above and stored in step 814 of FIG. 8(Self-Test/Calibration Processing), these photodiode output values areconverted to % absorbance values.

At step 908, peaks in the normalized absorbance data of 906 areidentified by applying a first derivative zero-crossing peak finder.Briefly, this involves calculating the first derivative of each point inthe ordered series (optionally employing smoothing coefficients, ifrequired for noise reduction) and identifying as peak maxima thosepoints whose derivatives:

1. have downward-going zero crossings;2. and have slopes exceeding a predetermined slope threshold, T_(S)3. at a point where the original signal's amplitude exceeds apredetermined amplitude threshold, T_(A).

The values of T_(A), T_(S), and the smoothing coefficients aredetermined empirically, based on observation of numerous testcartridges, to yield peak identifications conforming with trainedobservers' judgments and/or orthogonal test results such asenzyme-linked immunoassays (ELISA) of the same blood. As such, theseparameters are determined in advance and stored in memory of the readeras indicated at 909. These empirical observations benefit from ourability to test negative blood samples spiked with known amounts ofantibody, thus ensuring that our predetermined thresholds andcoefficient values do not exclude identification of weak true positivepeaks (to minimize false negatives) or include identification of weakstains as peaks (to minimize false positives).

At step 910, labels are assigned to the peaks defined in the abovesteps. The labels assigned are either as Control, IgM, or IgG peaks, orignored, provided they fall within predefined windows of scan positions(based on empirical observations of line locations in numerous testcartridges from several batch numbers). It should never be the case thattwo peaks are identified within one window. If this occurs, it isconsidered a cartridge error condition. If such an error condition isdetected it is reported as indicated at 912.

At step 914, the processing applies local baseline corrections to theControl, IgM and IgG peaks defined above (e.g., background subtraction)and calculates peak areas (same as area under the peak, AUP) A_(C),A_(M), and A_(G), respectively, treating the peaks as Gaussian in shape.Code examples are known in the art to persons of ordinary skill; for onein Matlab see, e.g.https://terpconnect.umd.edu/˜toh/spectrum/findpeaksb.m.

At step 916, the processing calculates test line peak to control linepeak ratios R_(M) and R_(G) as R_(M)=A_(M)/A_(C) and R_(G)=A_(G)/A_(C).These ratios are then used to report test results to the user, e.g., viathe display 34 or by the use of a reporting of a number on the display34 which has to be cross-referenced to instructions or literature forinterpretation. For example, in the COVID-19 scenario, ratios above anempirically determined preset threshold (again, determined in advanceand stored in memory) are considered ‘antibody-positive’ results for IgMor IgG, respectively. Alternatively, ratios falling within pre-definedranges may be classified as (e.g.) ‘high positive,’ ‘medium positive’,or ‘low positive’.

F. Positioning

As noted above, in both the calibration step and in the readingoperation the reader acquires a 1×N array of photodiode output values asa function of scan position. The photodiode output values for positionare obtained from the photodiodes 2904 in the assemblies 1110A and1110B, see FIG. 29.

This positional relationship between photodiode output and scan positionis accomplished by providing on the cartridge tray 32 a series of ruledregistration marks—thin black lines spaced at (e.g.) 1 mm (or,preferably 0.5 mm) intervals, hereinafter the “encoder strip” 1010, seeFIGS. 12, 13 and 25. In one possible arrangement, the encoder strip 1010can be produced as an adhesive sticker which is permanently affixed tothe top face of the carrier during manufacturing of the tray. To keepmanufacturing costs low, the positioning of the encoder strip on thetray need not be highly precise, e.g., requiring a jig or an alignmenttool during the carrier manufacturing process. Thus, the signalprocessing algorithm outlined below does not require precise knowledgeof the start- and end-points of the encoder strip with respect to theexpected positions of the test and control lines on the cartridge'snitrocellulose strip. Instead, it is sufficient that the sample encoderstrip merely spans the same length as the region of interest (ROI) ofthe nitrocellulose strip (the length spanned by the Control, IgM and IgGtest stripes) without spanning the ends of the nitrocellulose viewingwindow of the cartridge, and be reasonably parallel to thenitrocellulose strip 110 (FIG. 3). Optionally, a second encoder stripspans the length of the test pattern (without spanning the ends of thetest pattern sticker) and is reasonably parallel to it, as well. In oneembodiment these encoder strips are illuminated by LED(s) and read by asingle photodiode each, masked or otherwise having a field of viewencompassing only a single registration mark at a time as the carrier isinserted into the reader. This is the preferred arrangement shown inFIG. 11 at 1110A and 1110B.

Registering photodiode output with respect to position along either thenitrocellulose strip, or the test pattern, thus involves simultaneouslyreading the outputs from the sample photodiode (FIG. 4, 106) and theencoder photodiode 2904 of assemblies 1110A or 1110B (FIG. 11, 29).Thus, in the illustrated embodiment, the spacing of the encoder pattern1010 and its associated photodiodes in the assemblies 1110A and 1110B issuch that the calibration test pattern and test strip are read by thesample photodiode 106 at the same time as the encoder pattern is read bythe encoder photodiode in the assembly 1110A or 1110B.

The following procedure is then conducted:

1. A 2×N array of sample photodiode (106) output values and time-matchedencoder photodiode (1110A, 1110B) output values (hereinafter “the dataarray”) is stored in memory. Each element in this array comprises anequal time interval, i.e., equal to the temporal resolution of thesample photodiode's A/D converter.2. The amplitude discrimination peak finder algorithm of step 806,described above, is used to identify each registration line in theencoder photodiode's output.3. The total number of registration lines thus identified is counted. Ifthis number differs from the known total number of registration linesprinted on the encoder strip this constitutes an error condition—eitherthe carrier was not inserted all the way into the reader (typicallyyielding too few encoder lines identified) or else the carrier waspartially withdrawn from the reader at one or more points during fullinsertion (typically yielding too many encoder lines identified, as theencoder strip moves forwards, then backwards, then forwards again acrossthe photodiode's field of view during insertion).4. The encoder data is linearized with respect to time, in order tocorrect for possible variation in the speed at which the carrier wasinserted into the reader. This step comprises the following sub-steps:

a) Calculate the median and maximum peak widths of the detected encoderpeaks.

b) A maximum peak width greater than a specified threshold valueconstitutes a user error (the user interrupted the insertion process foran unacceptably long interval).

c) Adjust all encoder line peak widths to equal the median peak width,by applying the mathematical operations of erosion or dilation (erodethe too-wide peaks and dilate the too-narrow peaks to each equal themedian peak width). For an outline of erosion and dilation algorithmssee https://www.r-project.org/nosvn/pandoc/mmand.html. Ourimplementation will use a pre-defined kernel value [for example,(1,1,1)] empirically determined to give the optimal result.

d) Apply the same erosion or dilation functions to thetime-corresponding sample photodiode outputs.

e) Calculate the median and maximum inter-peak widths of the detectedencoder peaks.

f) A maximum inter-peak width greater than a specified threshold valueconstitutes a user error (the user interrupted the insertion process foran unacceptably long interval).

g) Adjust all encoder inter-peak widths to equal the median inter-peakwidth by deleting values from too-wide inter-peak regions or insertingadditional values into too-narrow inter-peak regions (the insertedvalues equal the mean of the two values immediately adjacent to theinserted cell).

h) Apply the same insertion or deletion operations to thetime-corresponding sample photodiode outputs.

G. Display

As shown in FIGS. 3 and 20, the reader includes a display 34 (which maytake any convenient form such as LCD or other), on which the results ofthe reading operation are displayed to a user. The display 34 could beprogrammed to display “positive” “negative” “error” or similarnomenclature, or could be programmed to display an alphanumeric indicia,such as “1”, “2”, “A”, “B” etc. which requires reference to externalinformation in order for a user to interpret the results. For example,such external information could take the form of a chart or the likeprinted on the top of the reader or in accompanying literature, such asshown in the Appendix of our prior provisional application.

H. Reader Electronics and Power Supply

The reader 20 includes electronics mounted to the board 1026 as shown inFIG. 10; such electronics could include a programmable processor orprocessing unit to perform calculations and generate results asexplained in this document and the Appendix of our prior provisionalapplication. Additionally, the electronics will include a memory storingparameters used in the calculations, as explained above, program code,etc. The electronics further include a rechargeable battery 1021 (FIG.10), a power supply to recharge the battery via the port 1702 of FIG.17, analog to digital converter(s), and additional conventionalcomponents the details of which are conventional and not particularlyimportant. Optionally, the reader includes a GPS unit; files of testresults include the GPS location of the reader, as shown in FIG. 41.

I. Interface to Remote Computing Devices

The electronics further includes a wireless transmitter 1027 (e.g.,Bluetooth, WIFI or the like) which serves to transmit test results fromthe reader to an external computing device, e.g., smartphone, laptop,desktop computer, etc. The test cartridge may further include a bar codeor RFID tag which is read within the reader in order to assign anidentity (e.g., name, or other identifier) to the test read.Accordingly, the reader 30 may further include a bar code reader or RFIDreader which reads the bar code or RFID tag before or during theoperation of reading the test device. The identifying indicia (name,employee number, etc.) is then associated with the test result and thecombination of test result and identifying indicia can be transmitted tothe external computing device, e.g., smart phone, using the wirelesstransmission capability of the reader, or optionally via the USB port onthe rear of the reader (FIG. 17), or in some other manner. In oneformat, this data is in the form of a CSV file.

It may also be desirable to upload anonymized test results to adatabase, either locally on the remote computing device or in computingplatform accessed over computer networks. In particular, the testresults and optionally information regarding the subject having the testperformed, such as age, sex, location, date/time, etc., but anonymized,is obtained from the reader or the reader in combination with a localcomputing device, e.g., smartphone, and transmitted to a web portal, ordatabase. This process can occur in a distributed manner, for exampleover a large number of readers deployed in a geographic area, such ascity, state or even country. The accumulation of such information in thedatabase can be mined for many possible beneficial uses, for example toidentify areas of high or low seroconversion rates.

Accordingly, in one possible configuration an app for a smartphone isprovided which includes features for receiving the test resultswirelessly from the reader 30 and additional user interface prompts orfeatures to enter information regarding the subject taking the test(optionally, their name, age, sex, location, time/date, and/or employeenumber, etc.) and such information is then transmitted to a database forstorage and possible mining.

As another possible configuration, the computing device (e.g.,smartphone) communicating with the reader wirelessly includes a GPS unitand an app or software that appends geographic coordinate locations tothe test results which are reported by the reader. The geographiccoordinates are determined by the computing device's GPS functionalityand indicate the location of the computing unit, but since the computingunit is local to the reader, the location information is essentially thelocation of the reader, and, by extension, the location (at least to anapproximate degree) of the person supplying the sample to the testdevice. In this configuration, the reader outputs the results and IDinformation (from bar codes, RFID tag or other) associated with theperson taking the test, and possibly other information such as peakdata, error codes, etc. and this information is passed by WIFI/Bluetoothto the computing device, e.g., smartphone. Embedded software in thereader's microprocessor controller generates an output CSV file withthis information. The computing device appends geographic coordinatedata (such as latitude and longitude) to the CSV file, such as shown inFIG. 41.

Alternatively, the GPS unit could be in the reader itself, or in theaccessory unit of FIG. 30 and described later in this document.

The CSV-formatted data may then be imported into any convenient localdata management application such as a spreadsheet or relationaldatabase, see FIG. 42, or uploaded to a cloud-based portal in ananonymized format, where it may be aggregated with the data obtainedfrom a multitude of other readers, potentially hundreds or eventhousands of readers. The aggregated cloud-based data can then be datamined, visualized, summarized or otherwise manipulated and presented togenerate reports, for example to spot trends in seroconversion rates orshow how seroconversion rates or test administration rates are trendingin particular geographic areas or among different demographic groups ofsubjects.

Because this epidemiological information includes geographic coordinateinformation, it may be visualized by being overlaid on a map to visuallydisplay geographic differences in the summarized data. Such overlays mayinclude heat maps or choropleth maps (both of which use color coding toindicate numeric differences over geographical areas), or a collectionof graphs overlaying a map. One possible example is shown in FIG. 43.This map overlays pie charts centered on each reader's geographiclocation to represent the frequency of positive test results by region(in this example, cities and/or counties).

J. Accessory Device with Programmable Movement and Repeated Reading ofTest Strip

In another possible configuration, an accessory device (see FIGS. 30-38and the description below) is provided which is used in conjunction withthe reader and cartridge of FIG. 3. The accessory device 3000 (FIG. 30)is designed to hold the reader 30 (FIGS. 31-32B) and provide anelectro-mechanical means or system for moving the tray 32 in and out ofthe reader in a programmed fashion to enable repeated readings of thetest cartridge over time. This means for moving the tray can take theform of a threaded rod 3302 (FIG. 33) or lead screw, which is activatedby a motor 3300 in the accessory device, and a pull 3304 or other deviceconnected to the threaded rod 3302. The pull 3304 engages mechanicallywith the tray to move it back and forth. Other suitable arrangementsthat function as described below can of course be devised.

Whereas in the embodiment of FIG. 3 the tray 32 and test cartridge 20 ismoved into the reader manually and a result is obtained virtuallyinstantly (e.g., in a few seconds), this accessory device configurationis designed for controlled, repeated reading of the entire exposedregion of the test strip, or portions thereof, for seconds or possiblyone or more minutes. The changes over time in the photodiode's detecteddecrease in test stripes' reflectance correlates to the gradual changesin intensity of the color change from lighter to deeper shades of red onthe test strip. This embodiment further enables monitoring and recordingthe elapsed time for emergence of a positive result on the test strip(i.e., a peak which meets the necessary characteristics for a positiveinterpretation using the procedure of FIG. 9). This elapsed time data,possibly including further information as to the peak (e.g., height),slope of the photodiode response, and/or ratio of the test peak to thecontrol line peak, can provide further information useful in assessingthe state of the sample and, therefore, the result generated by thereader. For example and without limitation, lower titers of targetantibody in the sample can give rise to more slowly-developing teststripe intensity, which can be detected by the photodiode output signalover time.

When a patient blood/buffer solution is applied to the sample cavity(22) in the cartridge 22 (FIG. 3), the sample progresses down the stripby capillary action. The leading edge of the sample, containingantigen-conjugated gold particles, moves further and engages thedetection zone, e.g., IgG (or IgM) stripe, and these bind and areimmobilized at the test stripe. Over time, the color grows more intenseas more antigen-conjugated gold binds there. Thus, as presentlyunderstood, a higher titer (i.e., concentration) of the antibodies ofinterest (e.g., COVID-19 antibodies, for sake of example) will lead tofaster emergence (and ultimately saturation) of the color in the IgGtest line. In this accessory embodiment, the movement of the cartridgeis controlled by the threaded rod and associated motor such that thephotodiode detector in the optics unit of the reader is positioned toread that IgG test line area repeatedly over time, e.g. over a period of1 to 60 seconds, or potentially longer (the time depending on manyfactors such as the test at issue). The movement of the tray may bepaused or stopped entirely during the reading of the test line. Themeasurements of the change in photodiode output, slope, final peakheight, etc., as a function of elapsed time, may be useful tocharacterize the titer of the patient's antibodies. In oneconfiguration, the optics unit of the reader is configured to repeatedlyscan the full test strip. Alternatively, the means for moving the traythen advances the tray and test cartridge further into the reader andthe photodiode detector then reads the IgM test line, optionally overtime as just stated as well, and then the Control line to record itscolor intensity, which will lead to an adjustment of the final readingresults for the IgG and IgM test line.

The movement of the cartridge relative to the optics unit and photodiodemay be programmed such that movement is stopped and the test line readrepeatedly in the sequence at which the sample moves down the test stripand contacts the test lines in order (the one closest to the sample wellbeing contacted first). It may be important, at least for some tests,that the reading of the test lines captures the initial ramp up, slopeand asymptotic leveling off of the peak intensity as the color changeoccurs in the test line over time, and that the cartridge be moved tothe second test line in the sequence in time to record that ramp up,slope and asymptotic levelling as well. These concerns would be obviatedif the optics unit of the reader is configured with a light source andreading unit that captures the reflectance of all the test stripeswithout necessarily having to move the test strip, such as by use of a2D imaging device or an array of photodiodes.

As an alternative it could be possible to place two optics units withinthe reader, one for the IgM line and one for the IgG line, so that boththe IgM and IgG lines are read independently and simultaneously by theoptics units over some period of time. Thus, measurements of the changein photodiode output, slope, final peak height, etc., as a function oftime, are made for both lines independently and simultaneously, and thatinformation can be processed within the reader to either generate a testresult (e.g., “positive”) or to characterize the result, e.g., “highpositive”, “low positive” or potentially “indeterminate.”

In this embodiment a QR code can be placed on the bottom surface of thecartridge 20, which is read by the accessory device.

Referring now to FIGS. 30-38, the accessory device 3000 includes acradle 3002 securely holding the reader 20. A drawer pull 3004 isconnected to the threaded rod, which slides relative to the cradle inslots 3006. The pull 3004 engages mechanically with the tray (see FIG.37) and guides the tray in and out of the reader. FIGS. 31 and 31A showsthe accessory 3000 with the reader 30 held in the cradle 3002 and themotor energized to extend the pull 3004 to a fully extended position inwhich the test device 20 is loaded into the tray 32. FIGS. 32 and 32Ashows the configuration where the motor is energized to insert the tray32 into the reader 30, e.g. during reading. FIG. 32B shows how the fitsinto the accessory unit by expanding slightly the cradle 3002 to allowit to snugly fit in place as show in FIGS. 31 and 32.

FIG. 33 is a perspective view of the electro-mechanical components inmore detail, such components being located in with the accessory unitper se. The pull 3004 is mounted to a fixture 3304 which slides on arail 3306. The motor 3300 rotates the threaded rod 3302 which causes thecollar 3308 mounted to the end of the rod to move towards or away fromthe motor 3300, and thereby cause the pull 3004 to move between thepositions shown in FIGS. 31 and 32.

The accessory includes a QR code reader 3310 which reads a QR code 3502on the bottom surface of the test device 20. The aperture 3312 in thepull enables the reader 3310 to read the code as the drawer and testdevice are moved into the reader.

FIG. 34 shows the motor 3300 energized to move the pull 3004 to theextended position, as in FIG. 31. FIG. 33 shows the motor energized tomove the pull 3004 to the closed position, as shown in FIG. 38. FIG. 34A is an exploded view of the electro-mechanical system and the accessoryunit 3000.

FIG. 35 shows the QR code 3502 on the bottom surface of the test stripwithin the cartridge 20. The tray 32 includes an aperture 3500 whichreveals the QR code when the cartridge is placed in the tray. The bottomof the reader 30 includes an aperture 3504 which reveals the QR code3502 when the tray is fully inserted so that it can be read by the QRcode reader 3310 (FIG. 33). See FIG. 36.

FIG. 37 is a perspective view of the electro-mechanical system andreader and tray from below, with the tray in the extended position,showing a slot 3700 in the end of the tray in which the pull 3004 fitsin order to engage the tray and allow it to be slid back and forth alongthe rail 3306.

FIG. 38 is a perspective view of the electro-mechanical system andreader and tray from below, with the tray moved to the closed or readingposition, showing the QR reader 3310 in position to read the QR code3502 placed on the bottom of the test device.

In one specific embodiment, an accessory unit 3000 to a lateral flowassay reader 30 having a moveable tray 32 holding a test device 20 hasbeen described. The tray is moveable from open and closed positions(FIGS. 37, 38). The accessory unit 3000 includes a structure holding thereader, e.g., the cradle of FIG. 30, and an electro-mechanical system(3300, 3302, 3304, 3004) engaging the tray 32 and moving the tray andtest device into the reader between the open and closed positions in aprogrammed manner (see FIGS. 37, 38), such that the test device ispositioned proximate to the optics unit 1028 in the reader (FIG. 10)such that at least one of the one or more test lines is readcontinuously for a period of time by the optics unit, whereby the readeris able to record changes in the color intensity of the one or more testlines over time. In one possible embodiment, the reader is as shown inFIGS. 10 and 4 and discussed previously; however variations from thatconfiguration are possible.

In one possible embodiment, the test device include at least two testlines (FIG. 3), and the programmable movement moves the tray such thatthe movement is stopped when both test lines are positioned proximate tothe optics unit enabling the at least two test lines to be readcontinuously for a period of time by the optics unit. Optionally, theoptics unit includes a photodiode reading the at least one test linesand a memory storing photodiode output as a function of time while themovement of the tray is in stopped condition. As shown in FIGS. 33 and38, the accessory unit 3000 includes a QR code reader 3310.

The energizing of the motor 3300 and thus movement of the tray+cartridgeinto the reader is programmably controlled by a suitable processor inthe accessory unit 3000 and program code for the motor written toaccomplish the functions as described above. Thus, the accessory unit3000 of FIGS. 30-38 includes a memory storing program code, one or moreprocessors or microprocessors, a timer or clock, and additionalelectronics (power supply, etc.) to operate as described, and suchdetails are of course within the ability of those skilled in the art ofelectro-mechanical systems. Optionally, the accessory unit includes aGPS unit.

Preferably, the QR code affixed to the bottom of the test device ispositioned in a predetermined position, i.e., the location necessarysuch that it is exposed to the QR reader 3310 in the accessory unit whenthe tray is inserted for reading. The QR code can be applied to the testdevice at the time of use, by means of including the QR code as aportion of a sticker or cardstock item, with peel off adhesive orbacking, to facilitate attachment of the sticker (and thus the QR code)in the correct location. For example, with reference to FIGS. 39 and 40,the sticker 4002 can include a guide 4000, or physical dimensions, suchthat the sides and one end of the sticker 4000 align with the sides andone end of the cartridge 20, thereby positioning the QR code 3502 thenecessary distance (e.g., 1 inch) from the end of the cartridge 20 suchthat it appears in the window 3504 as shown in FIGS. 35 and 38. Forexample, the sticker 4002 has a width substantially equal to the widthof the test device and the QR code 3502 is placed on the sticker adistance from one edge thereof (such as the end of the sticker) suchthat when the one end thereof is aligned with the end of the test deviceand the sticker affixed in this alignment position the QR code is in thepredetermined position as shown in FIGS. 35 and 38. Examples of theguide 4000 are shown in the FIGS. 39 and 40. The portion 4004 of thesticker 4002 can have an adhesive backing, as well as the back side ofthe QR code, such that when the guide 4000 is removed the portion 4002of the sticker also is removed only leaving the adhesive portion 4004and the QR code 3502. FIG. 40A shows the QR code on the bottom the testcartridge after the guide has been used in the manner described.

Features and Advantages

The reader of this disclosure provides a number of advantages andfeatures.

1. Ease of Use

The reader is designed to impose the least possible workflow burden onthe test administrator or user. The user simply slides the testcartridge 20 into the portable, battery-powered reader 30 and thenimmediately removes it, to record an instant readout.

Optionally, results are recorded in an industry-standard CSV file formatfor simple Bluetooth upload to a spreadsheet, a corporate database,and/or, after anonymization, to a managed web portal, thus obviating theusual need for manual recordkeeping during test administration. Accessto anonymized web portal data will also allow detailed data mining tomonitor actual test cartridge and reader performance in the field andevaluate use patterns—analyses that should prove invaluable foreverything from FDA approval to ex post facto quality assurance to newproduct development. It could also yield unparalleled andepidemiologically valuable insight into the evolution of the geographicdistribution and spread of disease, e.g., COVID-19 seropositivity, inreal time.

2. Reproducible Results.

The test reader's integrated optics and light source obviate therequirement for controlled lighting conditions in which to consistentlyread LFIA tests, producing consistent results in test environmentsranging from the most dimly lit room to the brightest sun-drenchedparking lot.

3. High Sensitivity and Accuracy.

Weak-positive LFIA results are notoriously difficult to readconsistently (“is that a pale red line, or just a dim stain?”). Thereader's signal-processing software accurately and sensitivelyidentifies and quantifies weak-positive test lines and rejects randomstains.

4. Eliminate Lot-to-Lot Test Variability.

A manufacturer of the disclosed reader 30 may largely at the mercy ofthe test device manufacturer's unknown quality control standards, whichmay or may not be up to regulatory expectations. The reader'sratiometric signal processing removes as much variance as possible byjudging test results as the ratio of test line intensity to control lineintensity on the same cartridge (I_(T)/I_(C)), helping to ensureconsistent results even in the face of reasonably wide lot-to-lot testvariability.

5. Rugged Reliability.

In one possible configuration, the reader has no moving parts, making itboth rugged and simple to manufacture at low cost. It's simple yetsophisticated self-test feature obviates the usual need for factorycalibration of each reader and enables the reader to automaticallycompensate for performance degradation over time, thus greatly extendingits useful lifetime even when used in harsh environmental conditions.

6. Security Features Employers Want.

The reader may be configured with an RFID reader for corporate use, toautomatically record each test subject's identity (via a no-touch readof the employee's RFID-enabled company ID badge), assuring that eachtest result is properly assigned to the correct employee by makingrecordkeeping errors nearly impossible. In addition, this feature canprovide a measure of fraud protection, making it difficult for a‘stand-in’ to impersonate another employee (for example, anantibody-positive employee impersonating an antibody-negative friend).

While presently preferred embodiments are described with particularity,variation from the details thereof is possible within the scope of thepresent invention. The appended claims are offered by way of furtherdescriptions of the disclosed subject matter.

We claim:
 1. A method for reading a lateral flow test device having oneor more test lines and a control line with a reader having electronicsand an optics unit and a tray extendable from the reader from an openposition in which the test device is placed in the tray to a closedposition whereby the test device is read by the optics unit, comprisingthe steps of: reading a calibration test pattern placed on the tray withthe optics unit, the optics unit including a photodiode; performing aself-test of the reader's electro-optical system, wherein the self-testincludes generating a calibration curve S characterizing any non-linearresponse of the electro-optical system; and reading the test and controllines of the test device with the optics unit, including using thecalibration curve S to convert photodiode output values to % absorbancevalues.
 2. The method of claim 1, wherein the calibration test patternis in the form of a linear series of bands of known, graded opticalintensities, each of the bands oriented perpendicular to the axis of thetest device.
 3. The method of claim 2, wherein the linear series ofbands takes the form of at least five bands of known graded opticalintensities.
 4. The method of claim 2, wherein the linear series ofbands takes the form of at least ten bands of known graded opticalintensities.
 6. The method of claim 2, wherein the calibration testpattern is printed on a material and the material is placed in adesignated location on an upper surface of the tray.
 7. The method ofclaim 1, wherein the optics unit comprises a diode laser generating alight output, a line generator converting the light output of the diodelaser to a line format, and the photodiode, the photodiode reading theintensity of the laser light reflected from the test device.
 8. Themethod of claim 7, wherein the line format of the laser light isoriented perpendicular to the axis of the test device and in the sameorientation of each of the bands of the calibration optical testpattern.
 9. The method of claim 1, wherein the test device comprises atest strip.
 10. The method of claim 1, wherein the test device comprisesa cartridge containing a test strip.
 11. The method of claim 1, whereinthe test device includes a test strip configured to test for presence ofantibodies to the SARS-CoV-2 virus.
 12. A method for calibrating areader for a lateral flow test device, the reader including an opticsunit including a photodiode receiving light reflected from a test stripprovided with the test device, comprising the steps of: a) scanning inthe reader a calibration test pattern; b) acquiring photodiode outputvalues during the scanning step a); c) optionally inverting the outputvalues; d) calculating average peak heights Ai in the output values; e)storing the results of step d) as an array of peak heights and linearposition data [Ai, Pi], the linear position data Pi indicative ofincremental linear positions at which the calibration test pattern wasscanned in step a); and f) fitting a spline curve S to the array dataand saving the equations for the spline curve S, wherein the spine curveS characterizes any non-linear response of the optics system orassociated electronics.
 13. The method of claim 12, wherein the scanningstep a) comprises manual insertion of a tray carrying the test deviceinto the reader, and wherein the tray includes the calibration testpattern.
 14. The method of claim 12, wherein the test device includes atest strip configured to test for presence of antibodies to theSARS-CoV-2 virus.
 15. The method of claim 12, wherein the calibrationtest pattern is in the form of a linear series of bands of known, gradedoptical intensities, each of the bands oriented perpendicular to a longaxis of the test device.
 16. The method of claim 12, wherein thecalibration test pattern is printed on a material and the materialaffixed to the tray in alignment with the axis of the test device.
 17. Amethod of reading a lateral flow test device with a reader, the readerincluding an optics unit including a photodiode receiving lightreflected from a test strip provided with the test device, comprisingthe steps of: a) scanning with the reader the test strip; b) acquiringphotodiode output values during the scanning step a); c) optionallyinverting the output values; d) converting the photodiode output valuesto % absorbance values using equations for a spline curve S, wherein thespine curve S characterizes any non-linear response of the optics unitor associated electronics, and wherein spline curve S is generatedduring calibration of the optics unit prior to scanning step a); e)identifying peaks in normalized absorbance data; f) assigning labels tothe peaks identified in step e); g) applying local baseline correctionsand calculating peak areas for each of the labeled peaks of step f); andh) calculating ratios of peak areas associated with test lines of thetest device to a control line of the test device.
 18. The method ofclaim 17, wherein the spline curve S is obtained in a calibration stepperformed immediately prior to the scanning step a).
 19. The method ofclaim 18, wherein the spline curve S is obtained from performing theprocedure of claim
 1. 20. The method of claim 17, wherein the scanningstep a) comprises manual insertion of a tray carrying the test deviceinto the reader.
 21. The method of claim 17, wherein the test deviceincludes a test strip configured to test for presence of antibodies tothe SARS-CoV-2 virus.
 22. The method of claim 1, further comprising thesteps of transmitting the results of reading the test device to a remotecomputing device.
 23. The method of claim 22, wherein the remotecomputing device is a smart phone.
 24. The method of claim 22, whereinthe remote computing devices anonymizes the results.
 25. The method ofclaim 24, further comprising the step of transmitting the anonymizedtest results to a mineable database.
 26. A tray holding a lateral flowtest device and configured for movement between open and insertedpositions relative to a reader for reading the test device, the readerfurther including a ratchet pawl and a spring, the tray comprising: anupper surface, the upper surface having features for receiving andholding the test device; a set of ratchet teeth formed on the tray,wherein the pawl and spring of the reader are positioned so as to biasthe pawl into engagement with the ratchet teeth; the tray furtherincludes a first feature disengaging the pawl from the set of ratchetteeth upon complete insertion of the tray into the reader and enablingthe tray to be withdrawn from the reader; wherein when the tray is inintermediate positions between the open and inserted position the pawl,spring and ratchet teeth prevent retraction of the tray from the reader.27. The tray of claim 26, wherein the upper surface of the tray furthercomprises a calibration test pattern.
 28. The tray of claim 26, whereinthe upper surface of the tray further comprises a encoder pattern.
 29. Atray configured to receive a lateral flow test device having an axis andfacilitate insertion of the test device into and out of a reader havingan optics unit, comprising: structure holding the test device in thetray, and an optical calibration pattern on the tray in alignment withthe axis of the test device.
 30. The tray of claim 29, furthercomprising an optical encoder affixed to the tray.
 31. The tray of 29,wherein the optical encoder is affixed to platform positioning theoptical encoder such that the optical encoder is moved adjacent to anoptical reader for the encoder within the reader when the tray is movedinto the reader.
 32. The tray of claim 29, wherein the calibration testpattern is printed and affixed to an upper surface of the tray in adesignated position on the tray proximate to and in alignment with theaxis of the test device.
 33. The tray of claim 32, wherein thecalibration test pattern comprises a series of bands of graduatedintensity oriented perpendicular to the axis of the test device.
 34. Thetray of claim 29, wherein the tray further comprise a structureactivating a first position switch within the reader to turn on theoptics unit when the tray is inserted into the reader and a secondposition switch within the reader to turn off the optics unit when thetray is withdrawn from the reader.
 35. The tray of 34, wherein thestructure comprises a ramp formed in the tray.
 36. An accessory to alateral flow assay reader having (1) a moveable tray holding a testdevice having one or more test lines and (2) an optics unit for readingthe test device, the tray moveable from open and closed positions, theaccessory unit comprising: a structure holding the reader, and anelectro-mechanical system engaging the tray and moving the tray and testdevice into the reader between the open and closed positions in aprogrammed manner such that the test device is positioned proximate tothe optics unit such that at least one of the one or more test lines isread repeatedly for a period of time by the optics unit; whereby thereader is able to record changes in the color intensity of the one ormore test lines over time.
 37. The accessory of claim 36, wherein thetest device comprises lateral flow test device having an axis and one ormore test lines and a control line oriented perpendicular to the axis,and wherein the reader comprises: a housing enclosing electronics andthe optics unit configured for reading one or more test lines and thecontrol line of the test device; wherein the tray is extendable from thehousing between a closed position and an open position, wherein the trayis adapted to receive the test device when the tray is extended to theopen position, and wherein when the test device is placed in the trayand the tray moved to a closed position the one or more test lines andcontrol line are read by the optics unit; and wherein the tray furtherincludes a surface, the surface provided with an optical calibrationtest pattern spaced from the test device and positioned in alignmentwith the axis of the test device, the test pattern facilitatingperformance of a self-test of the optics unit upon movement of the trayfrom the open position to the closed position and immediately before thetest device is read.
 38. The accessory of claim 37, wherein the testdevice include at least two test lines, and wherein the programmablemovement moves the tray such that both test lines are positionedproximate to the optics unit enabling the at least two test lines toread repeatedly for a period of time by the optics unit.
 39. Theaccessory of 37, wherein the optics unit includes a photodiode readingthe at least one test lines and a memory storing photodiode output as afunction of time while the movement of the tray is in stopped condition.40. The accessory of claim 36, wherein the accessory further comprises aQR code reader.
 41. The accessory of claim 40, wherein the QR codereader is positioned within the accessory and oriented such that it canread a QR code provided on the test device when the test device is heldin the accessory.
 42. A system, comprising, a lateral flow assay reader,a test device having a bottom surface, and the accessory of claim 36,and further comprising: a QR code reader in the accessory, and a stickercontaining a QR code, wherein the sticker is sized and shaped, orprovided with a guide, so as to enable a user to attach the sticker tothe bottom surface of the test device such that the QR code is placed ina predetermined position on the bottom surface of the test device. 43.The system of claim 42, wherein the reader has a bottom surface havingan aperture formed therein wherein the QR code is applied to the testdevice in a position in alignment with the aperture enabling reading ofthe QR code by the QR code reader within the accessory.
 44. The systemof claim 43, wherein the sticker has a width substantially equal to thewidth of the test device and wherein the QR code is placed on thesticker a distance from one edge thereof such that when the one endthereof is aligned with the end of the test device and affixed thereatthe QR code is in the predetermined position.